Many extracellular proteins with diverse functions contain domains similar to epidermal growth factor (EGF), a number of which have a consensus Asp/Asn, Asp/Asn, Asp*/Asn*, Tyr/Phe (where the asterisk denotes a beta-hydroxylated residue). These include the coagulation factors IX and X, proteins with two EGF-like domains, the first of which contains the consensus residues. The first EGF-like domain of human factor IX contains a calcium-binding site, which is believed to be responsible for one of the high-affinity sites detected in this protein. Similar results have been obtained for bovine factor X. We have now used protein engineering and 1H-NMR techniques to investigate the importance of individual consensus residues for ligand binding. Measurement of a calcium-dependent Tyr 69 shift in the isolated first EGF-like domain from human factor IX demonstrates that Asp 47, Asp 49, and Asp 64 are directly involved in this binding. Gln 50, whose importance has previously been overlooked, is also involved in this binding. Two mutations in this domain, Asp 47----Glu, and Asp 64----Asn, present in patients with haemophilia B, reduce calcium binding to the domain greater than 4-fold and greater than 1,000-fold, respectively. Furthermore, the defective calcium binding of Asn 64 can be partially rescued by the compensatory mutation Gln 50----Glu. This latter mutation, when introduced singly more than doubles the affinity of the domain for calcium. This study thus defines residues involved in a new type of calcium-binding site and provides strong circumstantial evidence for calcium-binding motifs in many extracellular proteins, including the developmentally important proteins of Drosophila, notch, delta and crumbs.